PROJECT SUMMARY/ABSTRACT Cell polarity is necessary to pattern tissues and is essential in stem cells to generate cellular diversity during development and homeostasis. Accordingly, polarity defects can drive developmental disorders and diseases, including cancer. Previous studies have elucidated molecular mechanisms that generate polarity and break symmetry in single cells. How these mechanisms are integrated to generate complex tissues remains poorly understood. The stomatal lineage in the Arabidopsis leaf epidermis is an ideal system to quantitatively determine how intrinsic cell polarity is transmitted to daughter cell asymmetry due to its well-characterized developmental trajectory, optical accessibility, and genetic tractability. In this proposal, I will utilize the stomatal lineage to 1) test how a novel microtubule-dependent inhibitory loop utilizes conserved logic to generate polarity in stem cells and 2) delineate polarity-dependent mechanisms that drive daughter cell asymmetry. To accomplish the proposed aims, I have developed novel in vivo tools to manipulate polarity in the stomatal lineage with high temporal and subcellular precision. In Aim 1, I will use targeted depletion, molecular genetics, and high-resolution imaging to test how mutual inhibition between microtubules and cortical polarity proteins generate a single polarity axis in multipotent stem cells. Additionally, I will use quantitative in vivo imaging to experimentally test how integrating an inhibitory circuit with a parallel MAPK-dependent positive feedback loop increases polarity robustness. In Aim 2, I will leverage the strengths of the stomatal lineage to determine how stem cell polarity regulates differential daughter cell size. By acutely manipulating polarity and tracking resulting daughter cell size, growth, and fate decisions using long-term time-lapse microscopy in vivo, I will directly test how polarity regulates downstream asymmetry. Furthermore, by identifying novel polarity regulators in the stomatal lineage, I will identify molecular pathways that link polarity to differential daughter growth. Completion of the proposed aims will test polarity models during multicellular development and will identify the mechanisms linking polarity to stem cell asymmetry. Elucidation of polarity mechanisms in an evolutionarily divergent species has broad implications for polarity models and may define synthetic strategies to correct polarity defects in human disease states.